NGF Signaling Exacerbates KOA Peripheral Hyperalgesia via the Increased TRPV1-Labeled Synovial Sensory Innervation in KOA Rats

Objectives Knee osteoarthritis (KOA) pain is caused by nociceptors, which are actually sensory nerve fiber endings that can detect stimuli to produce and transmit pain signals, and high levels of NGF in synovial tissue led to peripheral hyperalgesia in KOA. The purpose of this study is to investigate how sensory nerve fibers respond to the NGF/TrKA signal pathway and mediate the peripheral hyperalgesia in KOA rats. Methods Forty SD male rats were randomly divided into 4 groups: normal, KOA, KOA + NGF, and KOA + siRNA TrKA. KOA model rats were induced by anterior cruciate ligament transection (ACLT). Mechanical and cold withdrawal thresholds (MWT and CWT) were measured 4 times in each group. The synovial tissues were harvested on day 28, and the expressions of NGF, TrKA, TRPV1, IL-1β, and PGP9.5 were determined using western blot, qPCR, and immunofluorescence staining. The primary rat fibroblast-like synoviocytes (FLSs) and DRG cells were divided into 4 groups as in vivo. The expressions of NGF, TrKA, TRPV1, and CGRP in vitro were determined using western blot and qPCR. Results KOA and intra-articular injection with NGF protein increased both mRNA and protein levels, not only TRPV1, PGP 9.5, and IL-1β in the synovial tissue, but also TRPV1, PGP 9.5, and S100 in the DRG tissue, while above changes were partly reversed after siRNA TrKA intervention. Besides, siRNA TrKA could improve peripheral hyperalgesia and decreased the TRPV1 positive nerve fiber innervation in synovial tissue. The results in vitro were consistent with those in vivo. Conclusion This study showed the activation of the NGF/TrKA signaling pathway in KOA promoted the release of pain mediators, increased the innervation of sensory nerve fibers in the synovium, and worsened peripheral hyperalgesia. It also showed increased TRPV1 positive sensory innervation in KOA was mediated by NGF/TrKA signaling and exacerbated peripheral hyperalgesia.


Introduction
Knee osteoarthritis (KOA) is a common degenerative disease, and the infammation of synovial tissue triggers nerve changes causing peripheral hyperalgesia in KOA which has received increasing attention.Chronic pain, as the main symptom of KOA, signifcantly afects the quality of life of patients worldwide.To date, there are no disease-modifying drugs or treatments that can stop or reverse the KOA process.Anatomically, KOA pain is caused by nociceptors, which are actually sensory nerve fber endings that can detect stimuli to produce and transmit pain signals through dorsal root ganglia (DRG) to the posterior horn of the spinal cord, causing a cortical refex to produce pain response.Nevertheless, the complex pathologic mechanisms of sensory innervation involved in KOA pain need further elucidation.
Nerve growth factor (NGF) is considered closely related to infammatory pain and neuropathic pain.As a potential target for pain treatments [1], it regulates neuronal growth and development and is important for sensory nerve innervation [2,3].NGF initiates signal transduction through its primary functional receptor tropomyosin receptor kinase A (TrKA) [4,5].Te activation of NGF/TrkA recruits many signaling molecules that bind to the intracellularphosphorylated tyrosine residues within TrkA by means of Src homology domains.At least two proteins have been identifed that were recruited: phospholipase C gamma-1 (PLCc1) and phosphatidylinositol-3-kinase (PI3K) [6][7][8][9].Tese pathways eventually sensitize transient receptor potential vanilloid 1 (TRPV1) and cause changes in the fuidity of intracellular and extracellular Ca 2+ , resulting in increased sensitivity of tissues and organs [10].Te activation of NGF/TrkA also promotes the release of infammatory mediators such as interleukin-1β (IL-1β) and pain-related neuropeptides and activates nociceptors to aggravate KOA pain [11] (Figure 1(a)).In a word, the NGF/ TrKA signaling pathway can directly or indirectly regulate TRPV1 [12], which is considered closely related to infammatory pain and neuropathic pain [13][14][15][16], and might regulate TRPV1 highly expressed in knee synovium, increasing nociceptor sensitivity and aggravating neuropathic pain [17].
Frontier researchers are attempting to fnd the link between NGF, sensory innervation, and KOA pain.Tey found the increased innervation in the symptomatic KOA synovial tissue, while the innervation and pain were decreased after using the NGF monoclonal antibody [18][19][20].NGF monoclonal antibodies have demonstrated impressive analgesic efects; however, clinical studies have found it may cause unacceptable adverse efects such as rapidly progressive KOA, and therefore, intervention with TrKA, the main functional receptor for NGF, may have better prospects for application.
Does nerve fber sprouting change sensory innervation in the KOA synovium and aggravate KOA pain (Figure 1(b))?Based on the above background, the main purpose of this study is to observe whether synovial nerve fber sprouting aggravates KOA pain and how is it regulated by the NGF/TrKA signaling pathway.

In Vivo Animal Experimental Design and Ethics
Statement.Animal experiments were conducted in accordance with the Chinese Guidelines for Animal Care and Welfare, and this study protocol was approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine (No. A210101).
Forty 8-week-old SD male rats, weighing 180-220 g, were obtained from Vital River Animal Technology (Beijing, China).Te rats were raised in an SPF environment under a temperature of 18-22 °C, a humidity of 28%, and light for 12 h/day.Te rats were randomly divided into 4 groups at day 0 by using a randomized digital table: normal (n � 10), KOA (n � 10), KOA + NGF (n � 10), and KOA + siRNA TrKA (n � 10) after one-week adaptive feeding.Te sample size was calculated by ANOVA assuming that α � 0.05 and power � 0.80.Te experimental groups were established by the KOA model for the ACLT method [21], 3% pentobarbital sodium (100 mg/kg) was injected intraperitoneally for anesthesia, and 1 cm-long incision was made to expose and cut the anterior cruciate ligament and repeated at the other knee.Antibiotics (80,000 unit penicillin) were intramuscularly injected to prevent infection once a day, 3 days in total.On day 14, the KOA + NGF group was intra-articularly injected with recombinant human NGFβ, 2 μg/joint once every two days, 14 days in total (7 injections); the KOA + siRNA TrKA group was intraarticularly injected with siRNA TrKA, 100 pmol/joint every two days, 14 days in total (7 injections); the normal group and the KOA group were injected with the same volume of 0.9% saline as control.

Preparation and Stimulation of Primary Rat Fibroblast-
Like Synoviocytes (FLSs) In Vitro.Synovial tissue was extracted from 6 to 8 weeks old SD male rats, washed twice with PBS, cut into 2-3 mm 2 size, homogenized, digested in 0.2% type I collagenase, and incubated at 37 °C for 4 h, fltered, centrifuged, and resuspended.10 mL of 10% FBS and 1% penicillin-streptomycin solution were added to the Petri dish.Te medium was changed every two or three days.Te 3rd to 5th generations of FLS were used for experiments.FLS was randomly divided into normal, LPS, LPS + NGF, and LPS + siRNA TrKA groups.Te LPS group was intervened with normal medium containing 5 μg/mL LPS [22] for 24 h and then changed to a normal medium for 24 h.Te LPS + NGF group was frst treated with a normal medium containing 5 μg/mL LPS for 24 h and then changed to a normal medium containing 20 μM NGF-β for another 24 h [23].Te LPS + siRNA TrKA group was frst treated with a normal medium containing 5 μg/mL LPS for 24 h, then transfected with siRNA for 6 h, and changed to a normal medium for 18 h.Ten, the FLS and supernatant were collected separately.DRG was extracted from the same SD rats, washed twice with PBS, put into mixed digestive enzymes, digested at 37 °C for 1 h, fltered, centrifuged, and resuspended.Inoculation was performed in 35 mm Petri dishes, and 2 mL of the medium was added at 37 °C.DRG neurons were divided into 4 groups corresponding to FLS and were intervened with the corresponding supernatant of the FLS culture medium for 24 h.

Pain Treshold Test. MWT was measured by the von
Frey test (Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, BME-404), referring to the method of Chaplan et al. [24], and the results were expressed in grams (g).CWT was measured by using a temperatureadjustable metal plate system (35150-001, Ugo Basil SLR, Italy).Nociceptive responses to cold (4 ± 0.5 °C) stimuli in the hind paws and the results were expressed in seconds(s).

Western Blot.
All of the synovial tissue and L3-5 DRG tissues were harvested and lysed in RIPA, and the protein concentration was measured by the BCA protein assay kit.Protein samples were separated on 12% SDS-PAGE gels and then transferred to PVDF.PVDF was blocked with milk 2 h at room temperature, the primary antibody was added overnight at 4 °C, and the secondary antibody was added for 2 h.Te images of protein bands were collected and analyzed by using the ImageQuant LAS4000mini imaging system.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).
RNA was isolated from tissues and cells with Trizol, respectively.Reverse transcription was performed by using 5 × HiScript II qRT SuperMix.qPCR was performed using SYBR qPCR MasterMix on ABI PRISM 7300.Te primer sequences are shown in Table 1.

Histology, Immunofuorescence Labeling, and Histological
Analysis.Te harvested cartilages and synovial tissues were fxed in 4% paraformaldehyde for 24 h, then decalcifed in 10% EDTA, embedded in parafn, sectioned, and stained with HE and Safranin O-Fast green.L3-5 DRG tissues were fxed in 4% paraformaldehyde for 6 h, dehydrated, and sectioned.Te primary antibody was added and blocked, and then, the sections were incubated in the immunofuorescence antibody dilution bufer for 12 h at 4 °C and incubated with the secondary antibody for 2 h at room temperature.Nuclear staining was performed with DAPI and then observed with a fuorescence microscope.Tree sections were taken from each group to observe the changes in synovial tissues and DRG tissues.Image analysis was performed with ImageJ software.
2.9.Statistical Analysis.Data are presented as mean-± standard deviation.Statistical analysis was performed with Graphpad Prism 8.0 software (San Diego, CA, USA) via ANOVA and two-way ANOVA, and P < 0.05 (two tailed) was considered as statistically signifcant.

Te Results of ACLT Modeling.
First of all, we examined the modeling results.Te anatomical changes showed the cartilage in the KOA group was uneven, thin, and worn, these changes were even more in the KOA + NGF group, and these changes were improved in the KOA + siRNA TrKA group (Figure 2(b)).HE and Safranin O-Fast green staining showed the cartilage in the KOA group and KOA + NGF group was worn and ruptured, the trabecular bone was disordered compared with that in the normal group, and the cartilage changes in the KOA + siRNA TrKA group were less.Te synovial HE staining in the KOA group showed synovial cells proliferated, were arranged disorderly, and were infltrated with more infammatory cells compared with those in the normal group; these changes were improved in the KOA + siRNA TrKA group (Figure 2(c)).Te western blot results showed there was a signifcant increase in the protein expressions of NGF, TRPV1, IL-1β, and PGP9.5 in the KOA group compared with the normal group ( * P < 0.05); these protein expression trends were similar in the KOA + NGF group; the protein expressions decreased in diferent degrees in the KOA + siRNA TrKA group ( # P < 0.05) (Figures 3(c) and 3(d)).
Te qRT-PCR results showed there was a signifcant increase in the gene expressions of NGF, TrKA, TRPV1, IL-1β, and PGP9.5 in the KOA group ( * P < 0.05), and these gene expressions were even higher in KOA + NGF group; these gene expressions were signifcantly decreased in the KOA + siRNA TrKA group compared with those in the KOA group ( # P < 0.05) (Figure 3(e)).

NGF/TrKA
Increased TRPV1-Labeled Sensory Nerve Fibers in Synovial Tissues.Te color of silver staining in synovial tissues was deeper, and the positive area of nerve fbers increased in the KOA group than that in the normal group; the color was lighter, and the positive area was decreased in the KOA + siRNA TrKA group (Figure 4(a)).
Te immunohistochemical staining images of synovial tissues showed the positive expressions of PGP9.5, TrKA, and TRPV1 in the KOA group were higher than those in the normal group.Tis trend was more obvious in the KOA + NGF group, and these positive expressions decreased in the KOA + siRNA TrKA group (Figure 4(b)).
To observe the causes of NGF-induced peripheral hyperalgesia and whether they were regulated by the NGF/ TrKA signaling pathway, we performed immunofuorescence staining on synovial tissues.We observed the positional relationship among TrKA, TRPV1, and PGP 9.5 by immunofuorescence in the synovial tissues, the results showed that TrKA, TRPV1, and PGP9.5 were colocated in each group (Figure 4(c)).We also observed the positional relationship among NGF, TRPV1, and PGP 9.5, and the results showed NGF, TRPV1, and PGP9.5 were two-by-two colocalized in the synovial tissues in each group.

NGF/TrKA Induced the Upregulation of TRPV1 and
Neural Markers in DRG.We detected the protein and gene expressions of pain-related TRPV1 and pan-neural markers PGP 9.5 and S100 in DRG tissues.Tere was a signifcant increase in the protein expressions of TRPV1, PGP9.5, and S100 in the KOA group and KOA + NGF group compared with those in the normal group ( * P < 0.05), while the protein expressions of TRPV1 and S100 were signifcantly decreased in the KOA + siRNA TrKA group compared with those in the KOA group, and yet, there was no signifcant diference in the protein expression of PGP9.5 ( # P < 0.05) (Figures 5(a) and 5(b)).Tere was a signifcant increase in the gene expressions of TRPV1, PGP 9.5, and S100 in the KOA group and KOA + NGF group compared with those in the normal group ( * P < 0.05), while these gene expressions signifcantly decreased in the KOA + siRNA TrKA group ( # P < 0.05) (Figure 5

(c)).
Te immunofuorescence of NGF and TrKA in DRG tissues was also observed.Te fuorescence intensity of NGF and TrKA was increased in the KOA and KOA + NGF group compared with that in the normal group, and the fuorescence intensity decreased in the KOA + siRNA TrKA group ( # P < 0.05) (Figure 5(d)).NGF and TrKA were colocalized to β3-tubulin, which is also a neural marker (Figures 5(e) and 5(f )).

NGF/TrKA Involved in LPS-Induced TRPV1-Related
Changes in FLS.First, FLS was identifed by antivimentin immunostaining and HE staining before the experiment (Figure 6(a)).Next, we verifed the above results with FLS.Te results showed there was a signifcant increase in the protein expressions of NGF, TrKA, and TRPV1 in the LPS and LPS + NGF group ( * P < 0.05), and these protein expressions decreased at diferent degrees in the LPS + siRNA TrKA group ( # P < 0.05) (Figures 6(b) and 6(c)).Tere was also a signifcant increase in the gene expressions of NGF, TrKA, and TRPV1 in the LPS and LPS + NGF group ( * P < 0.05), and these gene expressions signifcantly decreased in the LPS + siRNA TrKA group ( # P < 0.05) (Figure 6(d)).

NGF/TrKA Afects the Expressions of TRPV1 and CGRP in DRG Neurons.
Similarly, DRG neurons were identifed by GFAP and β3-tubulin immunostaining before the experiment (Figure 6(e)).Te results showed there was a signifcant increase in the protein expressions of NGF, TRPV1, and CGRP in the LPS and LPS + NGF group compared with

Discussion
Chronic pain has been an important burden for KOA patients, especially in advanced KOA patients, and KOA patients typically live with this chronic pain for about 30 years after diagnosis [25].Tere is still a lack of understanding of synovial sensory nerve sprouting, which may play an important mediating role in peripheral hyperalgesia.Terefore, further research is needed, and it is also useful for clinical decision.Te purpose of this study is to observe that the Pain Research and Management peripheral hyperalgesia thresholds decreased in all rats by these three modeling methods.Tey also found nerve fber sprouting in KOA synovium and verifed that the ACLT method can better simulate the pathological changes in human KOA.
In this study, we found that the peripheral hyperalgesia thresholds decreased in the KOA and the KOA + NGF group, while these thresholds were improved after intraarticular injection of siRNA TrKA to inhibit the NGF/TrKA signaling pathway.Te protein and gene expressions of infammatory factors (e.g., IL-1β), pain mediators (e.g., NGF and TRPV1), and neural markers (e.g., PGP9.5) were increased in KOA synovial tissues, and these protein and gene expression changes increased even higher in the KOA + NGF group; however, the expressions of these factors decreased in the KOA + siRNA TrKA group.Ten, we found that the protein and gene expressions trends of TRPV1, PGP9.5, and S100 in DRG tissues were similar to those in synovial tissues.
Next, we verifed the synovial innervation of sensory nerve fbers by silver staining and immunohistochemistry staining.Silver staining is mainly for nerve fber density.PGP9.5 is a pan-neural marker that is present in all afferent and eferent nerve fbers, is specifc to nerve fbers, and is often used as an axonal marker.Te positive area of silver staining and PGP9.5 of immunohistochemistry staining were increased in the KOA and the KOA + NGF group compared with those in the normal group; it indicated nerve fber sprouting.Te positive area of silver staining and PGP9.5 immunohistochemistry were decreased in the KOA + siRNA TrKA group as expected.In further research, the NGF and TrKA immunofuorescence in synovial tissues colocalized with TRPV1 and PGP9.5.Meanwhile, the fuorescence intensity of TRPV1 and PGP9.5 in synovial tissues decreased in the KOA + siRNA TrKA group compared with that in the KOA group.It indicated that some of the newly sprouting nerve fbers were TRPV1-labeled, and this pathological process is regulated by TrKA.
Te fuorescence of NGF and TrKA was co-localized with β3-tubulin, respectively, in DRG tissues and increased in the KOA and the KOA + NGF group and decreased in the KOA + siRNA TrKA group.It indicated that NGF might intervene DRG by forming the NGF/TrKA complex and then transporting to DRG via cytokinesis, and this process was regulated by TrKA.
FLS and DRG neurons were divided into four groups correspondingly as in vivo.After induction of the FLS infammation model with LPS, the protein and gene expressions of NGF, TrKA, and TRPV1 in FLS were signifcantly increased in the LPS group, this increased expressive trend was further in the LPS + NGF group, and this increased expressive trend was partly reversed after intervened with siRNA TrKA.Next, we intervened the DRG neurons with the FLS supernatant to simulate the coculture with FLS.Te protein and gene expressions of NGF, TRPV1, and CGRP were signifcantly increased in the LPS group and LPS + NGF group compared with those in the normal group, and there were also signifcantly decreased protein and gene expressions of NGF, TRPV1, and CGRP in the LPS + siRNA TrKA group compared with those in the LPS group.Tis is similar to the results in vivo, demonstrating that infammation can activate the NGF/TrKA signaling pathway and promote the release of pain mediators, and this progress may be regulated by TrKA.
Kc et al. [27] found the degree of cartilage damage was signifcantly improved, but peripheral hyperalgesia was increased in the PKC-δ knockout KOA rats, and attributed this contradiction to the increased expressions of NGF, TrKA, and PGP9.5 in the synovium.Tey pointed out that the inhibition of PKC-δ perhaps activated the NGF/TrKA signaling pathway, which may further promote sensory nerve fber sprouting, increasing peripheral hyperalgesia.Tis result is similar to our study, the nerve fber innervation in KOA synovium was signifcantly increased, and it might be regulated by the NGF/TrKA signaling pathway.Peripheral hyperalgesia can be regulated by the NGF/TrKA signaling pathway.On the one hand, the activation of the NGF/ TrKA signaling pathway increased the expression of infammation and pain mediators, and on the other hand, the NGF/TrKA signaling pathway promoted the innervation in the synovium.
Zhang et al. [21] found the density of sensory nerves increased in all rats modeled by MIA, DMM, and ACLT, and the expressions of NGF were upregulated.Tey also suggested sensory nerve sprouting in the KOA synovium was strongly correlated with KOA itself rather than the causes of KOA.Neuropathic pain was also related to TrKA and TRPV1 [28]; using the TrKA antibody can reduce the innervation of nerve fbers [18].All of these fndings indicate nerve fber sprouting was correlated with the NGF/TrKA signaling pathway and perhaps not be limited to the synovial tissue but also spread to the subchondral bone.
Which kind of nerve fbers will deteriorate the peripheral hyperalgesia in KOA? Te results of this study showed that the fuorescence of TRPV1 was increased in the KOA synovium and the binding of NGF and TrKA upregulated TRPV1 positive sensory nerve fber sprouting.Tis sprouting possibly increased the sensing area of stimuli in the synovium and deteriorated the local pain.Te NGF/TrKA complex enters DRG and activates TRPV1 there and increases the excitability of DRG so that it is easily activated by noninjurious signals and forms peripheral hyperalgesia.In short, the high level of NGF increased the nerve fber innervation of synovium, especially TRPV1-labeled sensory nerve fbers, and deteriorated peripheral hyperalgesia.Te above pathological changes were regulated by TrKA so that they were partially reversed and peripheral hyperalgesia improved after inhibiting TrKA.
Clinical trials with NGF monoclonal antibodies, which broadly inhibit the function of NGF and were found to cause rapidly progressive KOA, were once called of.Research on its functional receptor TrKA may facilitate further development of the potential to inhibit NGF for the treatment of chronic pain and beneft KOA patients.

Conclusion
In conclusion, this study showed that the activation of the NGF/TrKA signaling pathway in KOA promoted the release of pain mediators and infammatory factors, increased the innervation of sensory nerve fbers in the synovium, and worsened peripheral hyperalgesia, and the above pathological changes could be partially reversed by inhibiting TrKA.Tere are [29] still some limitations in this study due to the research conditions, such as not being able to set TrKA knockout or overexpression group, and not being able to quantify sensory nerve sprouting, which will be further improved in future studies.

Figure 1 :
Figure 1: Relationship between NGF/TrKA signaling pathway and KOA peripheral hyperalgesia.Notes.(a) Te activation of NGF/TrkA promotes the release of pain-related neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P (SP) and activates TRPV1 on nociceptor terminal; NGF/TrKA complex is transported to DRG via cytokinesis.(b) Te NGF/TrKA complex promotes DRG sensitivity and causes increased expression of TRPV1, PGP9.5, and S100, and it may also cause peripheral sensory nerve fber sprouting.

Figure 2 :
Figure 2: Te protocol of animal experiment and the results of ACLT modeling.Notes.(a) Animal experimental protocol.(b) Te anatomical changes in the knee.(c) HE (20x, scale bar � 500 μm) and Safranin O-Fast green staining (20x, scale bar � 500 μm) of the cartilage and the HE staining of synovial tissues (40x, scale bar � 50 μm).

Figure 3 :Figure 4 :
Figure 3: NGF-induced peripheral hyperalgesia and related protein and gene expressions in synovial tissues.Notes.(a, b) Te peripheral hyperalgesia thresholds.(c, d) Te protein expressions in synovial tissues.(e) Te gene expressions in synovial tissues.* P < 0.05 between the normal group and the KOA group.# P < 0.05 between the KOA group and the KOA + siRNA TrKA group.